Clinical Significance of Multi-Drug Resistance Associated Protein and P-Glycoprotein in Patients with Bladder Cancer

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Vol. 157, 1260-1265,April 1997 Printed in U.S.A.

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CLINICAL SIGNIFICANCE OF MULTI-DRUG RESISTANCE ASSOCIATED PROTEIN AND P-GLYCOPROTEIN IN PATIENTS WITH BLADDER CANCER MASAYUKI NAKAGAWA,* AKIO EMOTO, NOBWOSHI NASU, TOSHIKATSU HANADA, MICHIHIKO KUWANO, SUSAN P. C. COLE AND YOSHIO NOMURA From the Department of Urology, Oita Medical University, Oita. and Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan, and Cancer Research Laboratories, Queen's University, Kingston, Canada

ABSTRACT

Purpose: The clinical significance of multi-drug resistant proteins, such as multi-drug resistance associated protein and P-glycoprotein, in terms of prognostic value was determined in patients with bladder cancer. Materials and Methods: The expression of multi-drug resistance associated protein and P-glycoprotein was investigated immunohistochemically before and after chemotherapy. The relationship between expression of these multi-drug resistant proteins and clinical outcome assessed by tumor recurrence rate, cystectomy rate and 5-year survival rate was also investigated in 33 patients with bladder cancer. Results: Before chemotherapy multi-drug resistance associated protein expression was observed in 1 of 28 patients (4%) while P-glycoprotein expression was observed in 22 of 33 (67%). Multi-drug resistance associated protein induction by chemotherapy was observed in 6 of 28 patients (21%), whereas P-glycoprotein induction was noted in 4 (14%). Multi-drug resistance associated protein in this disease is induced more frequently by high dose (more than 300 mg.) than low dose (less than 300 mg.) anthracyclines (p <0.01).Immunohistochemical analysis also revealed co-expression of multi-drug resistance associated protein and P-glycoprotein in 5 of 28 patients (18%) after chemotherapy. However, there was no significant correlation between positive P-glycoprotein expression before chemotherapy and clinical outcome. Conclusions: Multi-drug resistance associated protein as well as P-glycoprotein mediated multi-drug resistance may be induced after chemotherapy for bladder tumors. However, the presence of P-glycoprotein before chemotherapy does not predict clinical outcome in patients with bladder cancer. KEY WORDS:bladder neoplasms, glycoproteins,proteins Following exposure to chemotherapeutic agents, tumor cells can acquire resistance to structurally and functionally unrelated compounds, termed multi-drug resistance. A p pearance of multi-drug resistant cancer cells severely limits the effectiveness of chemotherapy for a variety of common malignancies. Despite the structural diversity of these compounds, over expression of 2 genes, multi-drug resistance gene 1 (MDRl) and multi-drug resistance (associated) protein, have been demonstrated independently to be capable of conferring a multi-drug resistant phenotype.1.2 Over expression of the MDRl gene, which encodes a 1,280 amino acid transmembrane phosphoglycoproteintermed P-glycoprotein, has been responsible at least partly for the multi-drug resistant phenotype in tumors. P-glycoprotein, an energy dependent drug efflux pump, is often associated with decreased drug accumulation in numerous cancer cell lines in culture selected by drug resistance.3 A similar drug resistance profile has been reported in cell lines selected in vitro that do not display increased P-glycoprotein levels. Cole et al reported over expression of a new transport protein, multi-drug resistance protein, in several doxorubicin resistant cell lines.4 Analysis of the predicted amino acid sequence of multi-drug resistance associated protein revealed structural motifs char-

acteristic of a member of the superfamily of adenosine triphosphate binding cassette transmembrane transporters, the same superfamily to which P-glycoprotein belongs.6 Transfection experiments confirmed the ability of multi-drug resistance associated protein to be sufficient to confer multidrug resistance.6 Over expression of this protein has been detected in resistant cell lines derived from a variety of tumor types and several clinical specimens.?-9 However, to our knowledge the clinical significance of multi-drug resistance associated protein has not been studied in detail. Bladder cancer, a common urogenital malignancy, will recur in the majority of patients. Various anticancer agents have been used to decrease recurrence in patients with bladder cancer. Anthracycline antibiotics, including doxorubicin and epirubicin, and a platinum containing anticancer agent, such as cispiatin, are frequently used to treat bladder cancer but the therapeutic effects are less than satisfactory.'*." Appearance of bladder cancer cells resistant to such therapeutic agents is expected to be involved at least partly in the disappointing efficacy of these therapeutic agents. Enhanced expression of P-glycoprotein or the MDRl gene and/or multidrug resistance associated protein has been observed in bladder cancer cells resistant to multiple agents in vitro as well as in v i ~ 0 . l ~ However, '~ clinical significance of these multiAccepted for publication Oetober 18, 1996. drug resistant proteins has not yet been determined in terms Supportedby grant-in-aidfrom Ministry of Education,Science and of prognostic value. It remains unknown whether chemotherCulture, Japan and Yasuda Memorial Anticancer Research Fund. * Requests for reprints:Departmentof Urology, Oita Medical Uni- apy could modulate expression of P-glycoprotein or multiversity, Hasama-cho, Oita 879-55, Japan. drug resistance associated protein in bladder cancer. We 1260

MULTI-DRUG RESISTANCE ASSOCIATED PROTEIN AND P-GLYCOPRO" EXPRESSION IN BLADDER CANCER TABLE1. Expression

1261

of multi-drug resistant protein and P-glycoprotein in patients with bladder cancer treated with doxorubicin or

epirubicin BefondA€ter Chemotherapy

Pt.- Sex- Age

Stage

No.

- M -58 2 - M -84 3 -M 4-F 5 - F 6 -M 7 -M 8 -M

-78 -64 -75 -68 -68 -62

9 - M -82 10 - M -71 11 - M -84 12 - M -81 13 - F -82 14 - M -41 15 - M -61 16 - M -75 17 - M -63 18 - M -57 19 - M -70 20 - M -66 21 - M -39 22 - M -69 23 - M -71 24 - M -66 25-M-61

Drug Dose (mg.)

Multi-Drug Resistant

P-Glycoprotein

Clinical Outcome Survival (mos.)

Recurrence

Cystectomy

Yes No No No No Yes Yes Yes

No No No No No No No Yes

Alive ( 150) Alive (126) Alive ( 126) Alive (126) Alive (126) Alive (119) Alive (118) Died of disease (102)

Yes Yes No Yes No No Yes Yes No No Yes Yes Yes Yes Yes Yes Yes

No No No No No No Yes No No No No No No No No Yes No

Alive (67) Alive (60) Alive (68) Alive (52) Alive (51) Alive (50) Alive (49) Alive (47) Alive (44) Alive (40) Alive (39) Alive (33) Alive (31) Died of other disease (30) Alive (28)

-

~

1

Grade

Doxorubiein treatment POSJpoS. Pos./pos. PosJpos. NegJneg. NegJpos. PosJpos. PosJpos. NegJpos.

T1 T1 T1 T1 T1 T1 T1 T1

2 2 1 2 1 2 2 2

660 180 180 180 180 660 180 660

NegJneg. NegJneg. NegJneg. NegJpos. NegJneg. NegJpos. NegJneg. Negheg.

T1 T1 T1 T1 T1 T1 T2 TI T1 T1 T1 T1 Ta T1 T2

2 2 2 2 2 2 3 3 2 2 2 2 1

480 560 300 780 780 780 300 440 300 300 300 300 300 620 300 300 300

Epirwbicin treatment NegJpos. POSJpoS. POSJpOS. NegJneg. Not determined NegJnot determined NegJpos. NegJpos. Not determined Pos Jnot determined Not determined PosJnot determined NegJneg. Pos./pos. POSJpoS. NegJneg. PosJnot determined Not determined PosJnot determined Not determined Neg Jneg. PosJpos. NegJpos. NegJneg. Pos Jpos. Negheg. PosJpos. Neg./pos. PosJpos. NegJpos. NegJneg. NegJneg. Negheg. NegJneg.

__ T2 T1

2

2 3 3

investigated the expression of multi-drug resistance associated protein and P-glycoprotein immunohistochemically before and after chemotherapy in bladder cancer specimens from 33 patients. Clinical outcome after a 5-year followup was also determined to evaluate clinical significance of multidrug resistance associated protein and/or P-glycoprotein in these patients. Use of doxorubicin or cisplatin for treatment of bladder tumors is discussed in relation to P-glycoprotein and/or multi-drug resistance associated protein expression in these patients. MATERIALS AND METHODS

We obtained surgical specimens from 33 patients with bladder cancer before and after chemotherapy. No patient received prior chemotherapy or radiation therapy. A total of 25 patients with superficial bladder cancer underwent transurethral resection followed by intravesical doxorubicin or epirubicin at a dose of 20 mg. in 20 ml. saline instilled through a urethral catheter (total dose 180 to 780 mg., mean 300, table 1).Eight patients with invasive or multiple bladder cancers received intra-arterial cisplatin (50 mg.) combined with radiation therapy twice a week for 3 or 6 weeks preoperatively (table 2). Multi-drug resistance associated protein and P-glycoprotein expressions were determined a t biopsy or transurethral resection of the primary tumor before and within 3 weeks &er discontinuation of chemotherapy. In some patients expressions were also determined using samples from recurrent tumors. Patients who were assessed as being tumor-free postoperatively were monitored every 3 months by cystoscopy, urine cytology and abdominal computerized tomography during followup (range 13 to 150 months, average 60.8). Clinical outcome was assessed by tumor recurrence, cystectomy and 5-year survival rates. The relationship between immunostaining of the multi-drug resistant proteins and recurrence or cystectomy rate was tested statistically with Fisher's exact test. The Kaplan-Meier curves for 5-year sunrival were analyzed with a generalized Wilcoxon test. A multi-drug resistance associated protein specific mono-

-

Alive . -.. (26) ,- -,

Alive(21)

clonal anti-human antibody (QCRL-3) and P-glycoprotein specific monoclonal Lntibody (C219)* were used to detect the expression of multi-drug resistance associated protein and P-glycoprotein in the tumor samples. The immunoglobulin fraction of normal mouse serum was used as a negative control. PNP20 etoposide resistant human prostatic cancer cells over expressing multi-drug resistance associated protein and MCF-7IAdr doxorubicin resistant human breast cancer cells over expressing P-glycoprotein were used as positive controls.15 After de-paranizing and hydrating the specimens, tissue sections were initially incubated in 3% hydrogen peroxide in distilled water to decrease endogenous peroxide activity and then they were rehydrated in phosphate buffered saline. The tissue sections were incubated with blocking buffer (10% rabbit serum in phosphate buffered saline) for 20 minutes followed by incubation for 1hour at room temperature with multi-drug resistance associated protein or P-glycoprotein specific monoclonal antibodies. After this incubation the staining was completed with the streptavidinbiotin-peroxidase complex according to manufacturer instructions, resulting in a brown stain on the cells positive for multi-drug resistance associated protein or P-glycoprotein. All slides were reviewed by 2 of us (N. N. and T. H.) without any prior knowledge of clinical outcome. Notation was made of any immunostaining of multi-drug resistance associated protein and P-glycoprotein. In addition, the location (nuclear and/or cytoplasm) and pattern (homogeneous and/or heterogeneous) of staining were documented. Immunostaining was evaluated in well preserved tumor portions away from cautery artifact, which can disrupt the staining pattern. RESULTS

Immunohistochemical staining of multi-drzg resistance associated protein a n d P-glycoprotein. Immunohistochemical staining with QCRL-3, an anti-multi-drug resistance associated protein monoclonal antibody, revealed positive expression of multi-drug resistance associated protein in bladder

* Centocor, Malvern, Pennsylvania.

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MULTI-DRUG RESISTANCE ASSOCIATED PROTEIN AND P-GLYCOPROTEIN EXPRESSION IN BLADDER CANCER

TABLE2. Expression

of

multi-drug resistant protein and P-glycopmtein in patients with bladder cancer treated with cisplatin and mdintion BeforelAfter Chemotherapy

Pt.-Sex-Age No. 1

-M

2 -M 3 -M 4-M 5 - F 6 -M 7 -M 8-M

-76 -79 -76 -62 -79 -68 -56 -62

stage

T3 T2 T1 T1

T2 T1 T2 T2

Grade

3 3 2

2 2 2 2 2

Clinical Outcome

Clsplatin Dose (mg.)

Multi-Drug Resistant Protein

P-Glyeoprotein

Recurrence

Cystectomy

Survival (mos.)

300 300 600 300 300 300 300 300

NegJneg. NegJneg. Negheg. Negheg. Negheg. Negheg. Negheg. NegJneg.

Pos. I p s . Pos. I p s .

Yes Yes Yes Yes Yes Yes Yes Yes

Yes Yes No No

Alive 160) Alive (48) Alive (45) Alive 136) Alive (311 Alive (27) Alive (27) Alive 113)

cancer tissues in 7 patients (tables 1 and 2). In all 7 patients the staining of multi-drug resistance associated protein was localized to the cytoplasm and the staining pattern was homogeneous. Positive P-glycoprotein staining of bladder cancer with C219,an anti-P-glycoprotein monoclonal antibody, was observed in 24 patients. The staining of P-glycoprotein was localized exclusively to the cytoplasm, and the staining pattern was homogeneous in 16 and heterogeneous in 8 of 24 patients (data not shown). Co-expression of multi-drug resistance associated protein and P-glycoprotein was observed in 5 of 20 patients treated with intravesical anthracycline. However, co-expression of both proteins was not observed in any patient treated with cisplatin and radiation therapy. Coexpression of multi-drug resistance associated protein and P-glycoprotein was noted in bladder cancer cells and adjacent normal bladder epithelial cells after intravesical anthracycline in some patients. Multi-drug resistance associated protein and P-glycoprotein expression before and after chemotherapy. We examined the expression of multidrug resistance associated protein and P-glycoprotein in 25 patients with superficial and 8 with invasive or multiple bladder cancers (tables 1 and 2). Multi-drug resistance associated protein expression was noted in 7 of 20 patients (35%)after and 1 of 20 before intravesical anthracycline treatment, while positive P-glycoprotein expression was noted in 16 (80%)of 20 and 16 of 25 (64%),respecbvely (table 1). Positive P-glycoprotein expression aRer combined cisplatin and radiation therapy was observed in all 8 patients with invasive or multiple bladder cancers, compared to 6 of 8 before treatment (table 2). However, no multi-drug resistance associated protein induction was noted in patients who had received combined cisplatin and radiation therapy. All 7 patients whose tumors were positive for multi-drug resistance associated protein expression after chemotherapy received intravesical anthracycline treatment (table 1). Of 28 patients P-glycoprotein and multidrug resistance associated protein induction after chemotherapy was observed in 4 and 6, respectively (tables 1 and 2). In some patients with recurrent tumors multi-drug resistance associated protein and P-glycoprotein expressions were determined longitudinally. However, consistency in protein status was not always maintained (data not shown). Correlation between dose of anticancer agent and induction of P-glycoprotein or multi-drug resistance associated protein. A total of 25 patients received intravesical doxorubicin or epirubicin a t a total dose of 180 to 780 mg. a h r surgery for primary tumors. To examine a correlation between dose of anticancer agent and induction of multi-drug resistance associated protein or P-glycoproteinwe compared the induction rates for these proteins in the low dose (300 mg. or less) versus high dose (more than 300 mg.) treatment groups in 20 patients. Multi-drug resistance associated protein induction was observed in 2 of 12 patients after low dose versus 4 of 8 after high dose anthracyclines. This difference was statistically signifcant (p <0.0096).P-glycoprotein induction was noted in 2 of 12 and 2 of 8 patients, respectively. However, the difference was not statistically significant. These results

Neg. I p s . Pos. I p s .

Pos.I p s . Pos. I p s . Neg. I p s . POS. I W S .

No No Yes

No

suggest that multi-drug resistance associated protein may be induced more frequently by high dose than low dose anthracyclines. Correlation between P-glycoprotein expression and clinical outcome. Positive P-glycoprotein expression before chemotherapy was noted in 22 of 33 patients (67%).Therefore, we investigated whether P-glycoprotein expression before chemotherapy predicts clinical outcome in patients with bladder cancer as assessed by tumor recurrence, cystectomy and 5-year survival rates. Of 22 patients with positive P-glycoprotein expression 16 had recurrent tumor compared to 8 of 11 with negative expression during a 5-year followup, and cystectomy was performed in 3 and 3, respectively. These results indicated no significant correlation between positive P-glycoprotein expression before chemotherapy and tumor recurrence or cystectomy rate. Furthermore, we could find no significant difference in 5-year survival rate between the positive and negative P-glycoprotein groups. These results suggest that P-glycoprotein expression before chemotherapy does not predict clinical outcome. We also examined the relationship between tumor grade and P-glycoprotein expression before chemotherapy. Positive P-glycoprotein expression before chemotherapy was noted in 2 of 3 patients (67%)with grade 1, 16 of 24 (67%) with grade 2 and 4 of 6 (67%) with grade 3 disease, respectively, and tumor recurred in 1 (33%),17 (71%)and 6 (loo%),respectively (tables 1 and 2). These results suggest that there is no significant correlation between grade of disease and P-glycoprotein expression before chemotherapy, and that tumor recurrence rate increases as a function of tumor grade. DISCUSSION

Over expression of the 170 ma. drug e m u pump P-glycoprotein was observed in many multi-drug resistant cell lines and some human tumors.3 P-glycoprotein over expression was noted in 33 to 75% of patients with human bladder cancer.12.16 In contrast, Nooter et a1 reported that bladder cancers and normal bladder tissues express low levels of multi-drug resistance associated protein messenger ribonucleic acid (mRNA).17 Selection of bladder cancer cells in culture by drug resistance to doxorubicin often induces multi-drug resistance associated protein over expression and decreased doxorubicin accumulation, and our recent study also indicates that etoposidddoxorubicin resistance in human prostatic cancer cells induces multi-drug resistance associated protein over expression and decreased drug accuenhanced expression of multi-drug rem u l a t i ~ n .l5~ Thus, ~. sistance associated protein as well as P-glycoprotein is closely associated with drug resistance to multiple agents, such as doxorubicin and epipodophyllotoxins. In light of these findings we investigated whether multi-drug resistance associated protein and/or P-glycoprotein is induced by cancer chemotherapy in patients with bladder cancer, whether there is any correlation between induction of multidrug resistance associated protein and/or P-glycoprotein and

MULTI-DRUG RESISTANCE ASSOCIATED PROTEIN AND P-GLYCOPROTEIN EXPRESSION IN BLADDER CANCER

1263

dose of anticancer agents, and whether multi-drug resistance ing the clinical relevance of this multi-drug resistant protein gssociated protein and/or P-glycoprotein predicts clinical out- in bladder cancer. come in patients with bladder cancer. We first determined We next investigated whether P-glycoprotein and/or multiexpression of P-glycoprotein or multi-drug resistance associ- drug resistance associated protein expression before chemoated protein in bladder tumors before and after treatment therapy predicts clinical outcome, such as tumor recurrence with anthracyclines or cisplatin and radiation. Expression of rate, cystectomy rate and 5-year survival rate in our paP-glycoprotein before chemotherapy (which may represent tients. However, our results indicated no significant correlaan intrinsic drug resistance) was observed in 22 of 33 pa- tion between positive P-glycoprotein expression before chetients (67%),while multi-drug resistance associated protein motherapy and clinical outcome. These results suggest that expression before chemotherapy was noted in only 1 of 28 P-glycoprotein expression before chemotherapy does not pre(4%). Of 28 patients induction of P-glycoprotein and multi- dict clinical outcome. We found no significant relationship drug resistance associated protein expression after treat- between tumor grade and clinical outcome. Consistent with ment (which may represent an acquired drug resistance) was our results, others reported that there was no significant observed in 4 and 6,respectively. These results suggest that relationship between P-glycoprotein staining and chemotherintrinsic drug resistance in bladder cancer may be mediated apeutic response or surviva1.16.26.26A recent study by Pu et primarily through P-glycoprotein rather than multi-drug re- al, using 88 clinical samples of bladder cancer, also demonsistance associated protein, whereas acquired drug resis- strated that MDRl expression status did not correlate with tance may be associated with multi-drug resistance associ- the chemotherapeutic response in systemic or intravesical ated protein and P-glycoprotein expression in patients with models, although approximately 70% of bladder tumors exbladder cancer. pressed P-glyc~protein.~~ Clifford et al observed that MDRl Multi-drug resistance associated protein andor P-glya~protein mRNA levels were significantly greater in poorly differentiexpression occurred in tumor cells and the adjacent normal ated high grade (grade 3) than in well and moderately difbladder tissue in some patients. Moreover, our study revealed ferentiated low grade (grades 1 and 2) tumors, although no coexpression of both proteins in 5 of 28 patients (18%)with evidence was found to implicate MDRl mRNA levels as an bladder cancer. Despite a similar selection procedure, the indicator of tumor recurrence or progression.28 Since they doxorubicin resistant bladder KK47 cell line co-expresses observed considerable (up to 63-fold) variation in MDRl exmulti-drug resistance associated protein gene and the MDRU pression among tumors, they suggested that inter-patient P-glycoprotein gene, while the doxorubicin resistant bladder variation in tumor MDRl expression may have a role in the T24 cell line over expresses P-glycoprotein or multi-drug determination of individual response to chemotherapy. Facresistance associated protein.14 Previously, Brock et a1 tors other than P-glycoprotein may also be associated with demonstrated that over expression of multi-drug resistance intrinsic chemoresistance in patients with bladder cancer. Expression of multi-drug resistance associated protein apassociated protein mRNA preceded MDRl/P-glycoprotein over expression in multi-drug resistant lung cancer cell lines peared to be enhanced in bladder tumors after treatment co-expressing both multi-drug resistant proteins.'* This with anthracyclines but not with cisplatin. Since multi-drug phenomenon of sequential co-over expression of 2 distinct resistance associated protein was discovered relatively remulti-drug resistant genes raises some interesting aspects on cently, the evaluation of its importance as a prognostic the emergence of drug resistant genes in cancer patients. marker or drug resistant marker in clinical tumors is not yet Using classic fluctuation analysis, multi-drug resistance is established.4 Clifford et a1 examined multi-drug resistance believed to be due to selection of preexisting mutants with a associated protein mRNA levels in 25 patients with bladder although cancer using a reverse-transcriptase polymerase chain reacspontaneous mutation rate of approximately induction of the MDRl gene has also been reported.lS tion based technique.29 However, they observed no apparent Sequential expression of different drug resistant proteins relationship between multi-drug resistance associated promay be necessary for tumor cells to acquire a multi-drug tein mRNA and the subsequent response to chemotherapy in resistant phenotype after chemotherapy. Further study patients uniformly treated with cisplatin, methotrexate and should elucidate how tumor cells might select preferential vinblastine or epirubicin, cisplatin and rnethotrexate. We over expression of P-glycoprotein or multi-drug resistance could not determine the prognostic value of multi-drug resistance associated protein in patients with bladder cancer beassociated protein in response to anticancer agents. In bladder tumors treated with anthracyclines (doxorubi- cause the expression rate before chemotherapy was too low cin or epirubicin), multi-drug resistance associated protein (l/28) to evaluate. In contrast, Norris et al recently demonwas induced more frequently by high dose (more than 300 strated a significant correlation between the levels of multimg.) rather than low dose (300 mg. or less) drug therapy. In drug resistance associated protein and amplification of contrast, there appeared to be a preferential increase in N-myc, a negative prognostic marker, in patients with neuP-glycoprotein expression compared to multi-drug resistance roblastoma.9 In addition, multi-drug resistance associated associated protein expression when tumors were treated with protein levels were sigruficantly enhanced in patients with cisplatin and radiation, although cisplatin is not a classic glioma after cancer chemotherapy.30 Further study should be MDRl associated agent. The P-glycoprotein encoding genes done to determine the prognostic value of multi-drug resisare over expressed in response to environmental stimuli, tance associated protein in patients with bladder cancer. such as heat shock, arsenate and hepatectomy in rodent and human cells in vitro as well as in viv0.20,21Treatment of human cancer cells with anticancer agents, such as anthracyCONCLUSIONS clines and cisplatin, or ultraviolet light radiation also enWe investigated the expression of 2 multi-drug resistant hances MDRl gene expression through activation of its promoter, suggesting that human MDRl is a stress inducible proteins, P-glycoprotein and multi-drug resistance associgene.22.23 Consistent with this hypothesis, P-glycoprotein ated protein, in patients with bladder cancer. Multi-drug levels were increased in bladder tumors treated with anthra- resistance may be induced by these 2 proteins aRer chemocyclines or cisplatin combined with radiation. Petrylak et a1 therapy but the presence of P-glycoproteinbefore chemotherobserved that an increase in the proportion of cells express- apy may not have prognostic value in patients with bladder mg P-glycoprotein occurs after a combination chemother- cancer. Beck et al presented a number of recommendations to apy program containing drugs known to select for detect P-glycoprotein associated multi-drug resistance in P-glycoprotein expression in vitro.24 Thus, determination clinical samples, and future studies with these recommended methods are to be considered to reevaluate the role of

1264

MULTI-DRUG RESISTANCE ASSOCIATED PROTEIN AND P-GLYCOPROTEIN EXPRESSION IN BLADDER CANCER

18. B m k , I., Hipfner, D. R., Nielsen, B. S., Jensen, P. B., Deeley, R. G., Cole, S. P. and Sehested, M.: Sequential cwxpression of the multidrug resistance genes MRP and m d r l and their prodpatients with bladder cancer.31 ucts in VP-l6(etoposide)-selected H69 small cell lung cancer cells. Cancer Res., 55: 459, 1995. 19. Jaffrezou, J. P., Chen, G., Duran, G. E., Kuhl, J.3. and Sikic, REFERENCES B. I.: Mutation rates and mechanisms of resistance to etopo1. Chen, C., Chin, J. E., Ueda, K, Clark, D. P., Pastan, I., side determined from fluctuation analysis. J. Natl. Cancer Gottesman, M. M. and Roninson, I. B.: Internal duplication Inst., 86: 1152, 1994. and homology with bacterial transport proteins in the m d r l 20. Chin. K V.. Tanaka, S., D a r l i n m n , G., Pastan, I. and (P-elvcoDrotein) eene from multidrug-resistant human cells. Gottesman,' M. M.: Heat shock and arsenite increase expresCei,-47; 381, 19i6. sion of the multidrug resistance (MDR1) gene in human renal 2. Gros. 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Cancer Res., 5 2 6175, 1992. I.J., Hsieh, C.-Y. and Lai, M.-K.: Expression of MDR-1 gene in 9. Noms, M. D., Bordow, S. B., Marshall, G. M., Haber, P. S., C o b , transitional ceU carcinoma and its correlation with chemotherS. L. and Haber, M.: Expression of the gene for multidrugapy response. J. Urol., 1 5 6 271, 1996. resistance-associated protein and outcome in patients with 28. Clifford, S. C., Thomas, D. J., Neal, D. E. and Lunec, J.: Inneuroblastoma. New Engl. J. Med., 334: 231, 1996. creased mdrl gene transcript levels in high-grade carcinoma 10. Herr, H. W., Laudone, P. and Whitmore, W. F., Jr.: An overview of the bladder determined by quantitative PCR-based assay. of intravesical therapy for superficial bladder tumors. J . Urol., Brit. J . Cancer, 6 9 680, 1994. 138: 1363, 1987. 29. Clifford, S. C., Neal, D. E. and Lunec, J.: Alterations in expres11. Sternberg, C. N., Yagoda, A., Scher, H. I., Watson, R. C., Herr, sion of the multidrug resistance-associated protein (MRP) H. W., Morse, M. J . , Sogani, P. C., Vaughan, E. D., Jr., Bander, eene in high-erade transitional cell carcinoma of the bladder. N. and Weiselberg, L. R.: M-VAC (methotrexate, vinblastine, kit. J. Cancer, 7 3 659, 1996. doxorubicin and cisplatin) for advanced transitional cell carci- 30. Abe, T., Mori. T.. Wakabavashi. Y.. Nakaaawa, M.. Akivama, S., noma of the urothelium. J. Urol., 139: 461, 1988. Deeley, R. G., Cole, S. P-. C., Koike, K, Kohno, K., Kuwano, M. 12. Naito, S., Sakamoto, N., Kotoh, S., Goto, K, Matsumoto, T. and and Hori, S.: Unpublished data. Kumazawa, J.: Correlation between the expression of 31. Beck, W. T., Grogan, T. M., Willman, C. L., Cordon-Cardo, C., P-glycoprotein and multidrug-resistant phenotype in transiParham, D. M., Kuttesch, J. F., Andreeff, M., Bates, S. E., tional cell carcinoma of the urinary tract. Eur. Urol., 2 2 158, Berard. C. W.. Bovett. J. M.. BroDhv. N. A,. Broxterman. H. J.. 1992. Chan, H. S., DalLn, W. S.,'Dieiel,"M., FoJo, A. T., Gascoyne, 13. Shinohara, N., Liebert, M., Wedemeyer, G., Chang, J. H. C. and R. D., Head, D., Houghton, P. J., Srivastava, D. K., Lehnert, Grossman, H. B.: Evaluation of multiple drug resistance in M., k i t h , C. P., Paietta, E., Pavelic, Z. P. and Weinstein, R.: human bladder cancer cell lines. J. Urol., 150: 505, 1993. Methods to detect P-glycoprotein-associatedmultidrug resis14. Hasegawa, S., Abe, T., Naito, S., Kotoh, S.,Kumazawa, J., tance in patients' tumors: consensus recommendations. CanHipfner, D. R., Deeley, R. G., Cole, S. P. and Kuwano, M.: cer Res., 6 6 3010, 1996. Expression of multidrug resistance-associated protein (MFW, MDRl and DNA topoisomerase 11 in human multidrugresistant bladder cancer cell lines. Brit. J. Cancer, 71: 907, EDITORIAL COMMENT 1995. The discovery of inducible protein pumps that transport chemo15. Tasaki, Y., Nakagawa, M., Ogata, J., Kiue, A., Tanimura, H., Kuwano, M. and Nomura, Y.: Reversal by a dihydropyxidine therapeutic drugs out of cancer cells has incited great hope that this derivative of non-P-glycoprotein-mediated multidrug resis- well-defined mechanism of drug resistance could be exploited for tance in etoposide-resistant human prostatic cancer cell line. assessing prognosis, tailoring therapy and devising innovative stratJ. Urol., 154: 1210, 1995. egies to circumvent drug resistance. Unfortunately, most of these 16. Park, J., Shinohara, N., Liebert, M., Noto, L., Flint, A. and hopes have been unrealized and bladder cancer appears to be no Grossman, H. B.: P-glycoprotein expression in bladder cancer. exception. The authors present data on 2 mechanisms of chemotherJ. Urol., 161: 43, 1994. apeutic drug resistance in transitional cell carcinoma involving the 17. Nooter, K., Westerman, A. M..Flens, M. J., Zaman, G. J. R., multi-drug resistance associated protein or the classical multi-drug Scheper, R. J., van Wingerden, K. E.,Burger, H., Oostrum, R., resistance (MDR1) protein, P-glycoprotein. Using a totally immunoBoersman, T., Sonneveld, P., Gratama, J. W., Kok, T., histochemical approach in 33 cases of primary transitional cell carEggermont, A. M. M.,Bosman, F. T. and Stoter,G.: Expression cinoma (25 superficial and 8 muscle invasive) we learn that de now of the multidrug resistance-associated protein (MRP) gene in expression of P-glycoprotein is relatively common, occurring in twohuman cancers. Clin. Canc. Res., 1: 1301, 1995. thirds of patients, while pretreatment multi-drug resistance protein

P-glycoprotein as well as multi-drug resistance associated protein in clinical development of multi-drug resistance in

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MULTI-DRUG RESISTANCE ASSOCIATED PROTEIN AND P-GLYCOPROTEIN EXPRESSION IN BLADDER CANCER expression is uncommon. While both are inducible after exposure to htravesical anthracycline a t high doses, P-glycoprotein (but not multi-drug resistance protein) is also induced by stressful stimuli, such as intra-arterial cisplatin plus radiation. However, despite these tantalizing findings the key point of this study is that neither p-glycoprotein nor multi-drug resistance protein expression predicts clinical outcome to the very drugs, anthracyclines, for which they have been shown to transport in vitro. In this respect the authors join a chorus of other researchers in substantiating this observation. How does one reconcile this disappointing disparity between theory and practice? While a quantitative approach designed to measure intracellular drug concentration might provide some insight, it is

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equally if not more plausible that inherent tumor resistance to anthracyclines is due t~ more complex factors, such as heterogeneous clones, cell cycle variations, bystander protection, repair systems and so forth. Simply, variations in intracellular drug concentration cannot alone account for intrinsic limitations in drug efiicacy although it can make marginal drugs even less effective. Michael A. O'Donnell Division of Urology Beth Israel Deaconess Medical Center Harvard Medical School Boston, Massachusetts